The objective of Project 3 in this PPG is to exploit a microfluidic diagnostics toolbox established by our research team for quantification of multiple signaling events and genomic lesions from fine needle aspirated (FNA) biopsies or circulating melanoma cells (CMCs). We will examine the feasibility of applying minimally invasive sampling techniques (i.e., FNA biopsy and peripheral blood draws for CMC enrichment) to repeatedly sample melanoma cells over the course of BRAF inhibitor (BRAFi) treatment. Tumor cells isolated from FNA biopsies and CMCs then will be subjected to single-cell signaling profiling technologies including microfluidic image cytometry (MIC) for quantitative proteomic analysis of multiple signaling molecules, and the Fluidigm BioMark^'^ system for reverse-transcriptase polymerase chain reaction (RTPCR) and targeted DNA sequencing. With bioinformatic analysis, our microfluidic diagnostics enable a systems pathology approach, capable of dissecting tumor heterogeneity and monitoring temporal disease evolution. Our long-term goal is eariy clinical detection of resistance mechanisms, and 'in patient-treatment' based prediction of tumor responsiveness to articular kinase inhibitors based on signaling responses. Activating BRAF^(R)^ kinase mutations occur in 50% of human melanomas. Clinical experience with the novel mutant BRAF-selectlve inhibitor vemurafenib found an unprecedented 60-80% antitumor response rate among patients with BRAF^^^-positive melanomas. However, acquired drug resistance frequently develops after initial responses in almost all treated patients. Recent studies by our joint team found that mechanisms of acquired resistance to BRAF inhibition include reactivation of the MAPK pathway (e.g., via NRAS mutation) or activation of alternative signaling through the RTK/AKT pathway (e.g., via PDGFRp overexpression). To overcome BRAFi resistance, we need to better understand, monitor and study evolution of resistance mechanisms during BRAFi treatment. Project 3 aims to demonstrate microfluidic diagnostics for dynamic monitoring the clinical evolution of BRAFi resistance. As the joint research endeavor unfolds, our microfluldlcs-derived single-cell proteomic and genomic assays will be applied to detect the resistance-associated genomic and phospho-profile findings from Projects 1 and 2 in clinical patient samples to help guide therapy choices. We also envision that the proposed microfluidic diagnostics can be employed to assess that the Impact of BRAF inhibitors on immune therapies (Project 4). RELEVANCE (See instructions): A key issue In analyzing acquired resistance in melanoma is the limitation of repeated diagnostic measurements of tumors. This can be overcome by applying minimally invasive sampling techniques to characterize the progressive tumors over the course of treatment. The objective of Project 3 in this PPG is to exploit a microfluidic diagnostics toolbox for quantification of multiple signaling events and genomic lesions from fine needle aspirated (FNA) biopsies or circulating melanoma cells (CMCs).